Protein Kinase C Signaling and Pluripotent Stem Cell

描述(申请人提供)：多能干细胞具有发展成不同细胞类型的巨大潜力。然而，赋予干细胞维持多能性或分化为其他类型细胞的能力的分子机制尚不清楚。我们发现，一种单一的、选择性的蛋白激酶C(PKC)抑制剂对蛋白激酶C(PKC)亚型的抑制保持了小鼠胚胎干细胞(ES)的未分化表型，而不影响其多分化潜能。此外，抑制PKC亚型允许从小鼠囊胚中获得具有生殖线能力的ES细胞，并促进小鼠胚胎成纤维细胞向诱导的多能干细胞重新编程。这一探索性和发展性研究方案的目的是进一步验证该方法的有效性，在该方法中，PKC信号通路将被靶向从大鼠获得、增殖和维持新的ES细胞，维持人类ES细胞的多能性，并了解相关的分子机制。我们将在这项提案中设定两个具体目标。在第一个特定目标中，我们将检验这一假设，即PKC信号是决定哺乳动物ES细胞维持多能性与分化的重要途径，因此，抑制PKC亚型功能将有助于在不影响其完整发育潜力的情况下获得、繁殖和维持多能性干细胞。我们将测试用PKC抑制剂衍生和繁殖的大鼠ES细胞的体内发育潜力，方法是将它们注射到胚泡中产生嵌合体，并评估这些嵌合体的生殖系传播。此外，我们将确定PKC抑制是否保持了人类ES细胞的未分化表型，并将通过在免疫受损的小鼠中发展畸胎瘤来评估多能性。在第二个特定目标中，我们将确定与PKC抑制介导的维持干细胞多能性相关的分子机制。我们将测试特定的PKC亚型/S的功能是否在诱导小鼠ES细胞分化中起重要作用。此外，我们还将确定抑制ES细胞中的PKC亚型是否保持多潜能干细胞特有的染色质结构。我们预测，这项提案的成功完成将开辟新的研究领域，针对干细胞生物学和再生医学目的的PKC信号通路。 与公共卫生相关：多能干细胞能够分化为我们身体的其他细胞类型，这表明了医学科学的一个巨大潜力领域，即可以从干细胞产生组织特异性细胞用于再生医学目的。因此，了解维持干细胞多能状态并决定其分化为特定细胞谱系的分子机制和策略是再生医学非常活跃的研究领域。从这些研究中收集的信息将有助于我们理解维持ES细胞的多潜能状态并决定其向其他细胞系分化的分子机制。

DESCRIPTION (provided by applicant): Pluripotent stem cells have tremendous potential to develop into different cell types. However, molecular mechanisms that endow stem cells with the capacity to maintain pluripotency or to differentiate into other cell types are not well understood. We found that inhibition of protein kinase C (PKC) isoforms by a single, selective PKC inhibitor maintains the undifferentiated phenotype of mouse embryonic stem (ES) cells without affecting their multidifferentiation potency. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ES cells from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts towards induced pluripotent stem cells. The objective of this exploratory and developmental research proposal is to further validate the efficacy of the approach, in which PKC signaling pathway will be targeted to derive, propagate and maintain new ES cells from rat, to maintain pluripotency of human ES cells, and to understand associated molecular mechanisms. We will institute two specific aims in this proposal. In the first Specific Aim we will test the hypothesis that PKC signaling is an important pathway to dictate maintenance of pluripotency vs. differentiation of mammalian ES cells and, therefore, inhibition of PKC isoform function will help to derive, propagate and maintain pluripotent stem cells without affecting their complete developmental potential. We will test in vivo developmental potential of rat ES cells, derived and propagated with PKC inhibitors, by injecting them into the blastocysts to generate chimera and by assessing those chimeras for germline transmission. In addition, we will determine whether PKC inhibition maintains undifferentiated phenotypes of human ES cells and will assess pluripotency by developing teratomas in immune-compromised mice. In the second Specific Aim we will determine molecular mechanisms that are associated with PKC inhibition-mediated maintenance of stem cell pluripotency. We will test whether function of specific PKC isoform/s is important to induce mouse ES cell differentiation. In addition, we will also determine whether inhibition of PKC isoforms in ES cells maintains pluripotent stem cell-specific chromatin structure. We predict successful completion of this proposal will open up new areas of research to target PKC signaling pathway for stem cell biology and regenerative medicine purposes. PUBLIC HEALTH RELEVANCE: The ability of pluripotent stem cells to differentiate into other cell types of our body indicates a tremendously potential area of medical science, in which tissue-specific cells can be generated from stem cells for regenerative medicine purposes. Thus, understanding the molecular mechanisms and strategies that maintain the pluripotent state of stem cells and dictates its differentiation to a particular cell lineage is an area of very active research for regenerative medicine. Information gleaned from these studies will contribute to our understanding regarding the molecular mechanisms that maintain the pluripotent state of ES cells and dictates its differentiation to other cell lineages.